Apicomplexan parasites such as Toxoplasma gondii, the causative agent of toxoplasmosis, and Plasmodium falciparum, the causative agent of severe human malaria, are obligate intracellular pathogens. Repeated cycles of host cell invasion, parasite multiplication and host cell lysis are central to the pathogenicity of these parasites, yet little is known about the mechanisms of host cell invasion or the parasite proteins that mediate the process. One protein that has been the focus of intense interest is AMA1. AMA1 is conserved among apicomplexans, and Plasmodium AMA1 is a leading malaria vaccine candidate. AMA1 is released onto the parasite surface during interaction with host cells, where it is thought to play an essential - though still undefined - role in host cell invasion. The T. gondii homolog of AMA1 (TgAMAl) has recently been identified, and a new system for conditional gene expression in T. gondii has enabled the creation of a parasite line in which expression of TgAMAl can be experimentally manipulated. A decrease in TgAMAl expression in these parasites causes a significant decrease in their invasiveness. The conditional knockdown parasites provide an unprecedented opportunity to study the function of a model AMA1 protein. Specific Aim 1 will use the conditional knockdown parasites and the wealth of assays available in T. gondii to deter mine the specific role played by TgAMAl in invasion. Specific Aim 2 will explore the functional significance of proteolytic cleavage and release of TgAMAl from the parasite surface during invasion. Specific Aim 3 will characterize and determine the function of TgAMA2, an AMAl-like sequence that is also expressed in T. gondii tachyzoites. T. gondii causes life-threatening disease in the congenitally-infected fetus and in immunocompromised persons, including those with AIDS or Hodgkins' disease, and those undergoing immunosuppressive or cancer chemotherapy. Given the apparent importance of AMA1 in invasion and the central role invasion plays in pathogenesis, a greater understanding of the function of AMA1 and AMA1-related proteins will likely contribute to the development of new vaccine-based or chemotherapeutic approaches to preventing or controlling infection by T. gondii and other apicomplexan parasites.